The present invention, in some embodiments thereof, relates to TNF alpha convertase inhibitors (TACE inhibitors) for the treatment of inflammatory disorders including Crohn's disease and rheumatoid arthritis.
Members of the ADAM family have been recognized as major ectodomain shedding proteinases. ADAMs are mostly type I transmembrane proteins featuring a multi-domain structure consist of pro-domain, a catalytic domain, a disintegrin domain, an EGF-like (cysteine-rich) domain, a single transmembrane domain and a cytoplasmic portion (FIG. 1). The pro-domain inhibits ADAM proteinase activity by binding the catalytic Zn(II) while mediating specific protein-protein interactions with the enzyme active site surface. This domain is cleaved by another proteinase either intra or extra cellular to regain enzyme activity. ADAM disintegrin-domains can, in some cases, interact with integrins to influence cell adhesion and cell-cell interactions. The disintegrin- and/or EGF-like-domains are thought to be involved in substrate recognition and/or (hetero)-dimerization. The transmembrane region is followed by a C-terminus cytoplasmic region, which contains an SH3-binding motif that interacts with intracellular signaling proteins.
Up to date most of the examined shedding activity on the cell surface is mediated by ADAM17 and its close relative ADAM10. A large number of proteins were identified as substrates for ADAM17 including key immuno-regulatory cytokines like TNF-α, IL-6 and their receptors TNFR and IL-6R, cancer related molecule such as ErbB ligands and their receptors, signaling molecule as Notch-1, cell adhesion molecules such as L-selectin (CD62L) or ICAM-1 and even amyloid precursor protein. Since ADAM17 had been identified as the primary sheddase for TNF-α it was named the TNF-α converting enzyme—TACE. Hence large variable biological functions are linked to ADAM17, such as development, axon guidance, cell-cell interactions, signaling and its main role in releasing TNF-α in inflammation.
Despite the remarkably large number of ADAM17 substrates identified during the last decade, most of the proposed pathological roles of this metalloproteinase are related to just a few substrates including pro-TNF-α, the ligands of the EGFR and the Amyloid Precursor Protein (APP) (FIG. 2). Given the central role of these molecules in many biological processes, the number of diseases in which ADAM17 has been suggested as a therapeutic target has grown exponentially in the last years. Many compounds belonging to different chemical classes have been synthesized as selective TACE inhibitors. However, the tested small molecules have been withdrawn from Phase II clinical trials for rheumatoid arthritis because of lack of efficacy, specificity and hepatotoxicity in vivo [DasGupta, S., et al., Bioorg Med Chem, 2009. 17(2): p. 444-59]. This result is not surprising. The use of synthetic small molecule inhibitors suffers from a lack of specificity. The small molecule inhibitors targeted the active site of TACE, which is highly conserved among all members of the metazincin family (FIG. 3). Therefore, many unpredicted side-effects were encountered by unwanted cross inhibition [Moss, M. L., et al Nat Clin Pract Rheumatol, 2008. 4(6): p. 300-9].
Inhibition in vivo is achieved by the naturally occurring endogenous MMP inhibitors, the Tissue Inhibitors of Metalloproteinases family (TIMP). This family consists of 4 members, namely TIMP1-4, which exhibit high sequence homology. Under normal physiological conditions, the activity of MMPs is mostly regulated by the TIMPs. Misregulation can lead to arthritis, cancer, multiple sclerosis and cardiovascular disease. Interestingly, the N-terminal domains can fold independently and fully inhibit the activity of the targeted MMPs or ADAMs. The inhibition mechanism involves a direct coordination of the catalytic Zn(II) via an N-terminal cysteine of the TIMPs as well as protein-protein interactions with the enzyme extended catalytic cleft. This confirms the mechanistic importance of metal chelation by the N-terminal amino group in metalloproteinase inhibitory activity. The selectivity towards a targeted MMP is achieved by four residues in the N-terminus populating the catalytic cleft by backbone contacts mimicking a binding of a substrate. Interestingly, an overall topology view reveals a “lock and key” protein-protein interactions that take place between the surface residues of the MMPs and the TIMPs (FIGS. 4A-4B). TACE is naturally inhibited by TIMP-3 as shown in the crystal structure by Wisniewska et al 2008 [ J Mol Biol, 2008. 381(5): p. 1307-19].
Similar to other members of the MMP family, TACE is generated as a latent zymogen and is activated upon the release of the inhibitory pro-domain. The activation of TACE zymogen is performed mainly by Furin-like protease, a proprotein convertase, in the late Golgi compartment through a Furin consensus site in the junction between pro-domain and the catalytic-domain 211RVKR214/R215. It has been found that the TACE pro-domain is essential for secretion of this enzyme in a functional form. TACE expression constructs that lack the pro-domain failed to secrete as a functional enzyme in insect cells and the protein retained in the endoplasmic reticulum and subject to degradation. It was concluded that the TACE pro-domain work as an intramolecular chaperone, assisting in the secretion of this proteinase.
The cytokine TNF-α plays a major role in autoimmune disease processes in inflammatory bowel disease and rheumatoid arthritis. Therefore, TNF-α has become an important target for the development of therapeutic strategies for the treatment of Crohn's disease and rheumatoid arthritis. TNF-α inhibitors that have been approved for clinical use to treat autoimmune disorders are infliximab, adalimumab and etanercept. Infliximab and adalimumab are modified antibodies specific for TNF-α whereas, Etanercept is a fusion protein comprising the ligand-binding portion of the human p75 TNF receptor (TNFRII) and the Fc fragment of human IgG1. The TNF-α inhibitors block the interaction of TNF-α with cell-surface receptors (FIG. 5).
A different strategy, highlighted by the design of small molecule inhibitors, is to prevent the release of soluble TNF-α from its membrane bound precursor. TACE being the major sheddase for TNF-α in immune-competent cells became a rationale target for this strategy. Orally available small molecule inhibitors were developed and demonstrate improved selectivity for TACE over the closely related matrix metalloproteinases [Georgiadis, D. and A. Yiotakis, Bioorg Med Chem, 2008. 16(19): p. 8781-94]. However, all of them failed due to liver toxicity caused by the unspecific side-effects of cross inhibition of other members of the MMP family.
Use of the pro-domain to inhibit TACE has been disclosed in Leonard, J. D., et al. Biochem J, 2005. 387(Pt 3): p. 797-805; Gonzales, P. E., et al., J Biol Chem, 2004. 279(30): p. 31638-45; Maskos, K., et al. Proc Natl Acad Sci USA, 1998. 95(7): p. 3408-12; Li et al., Int J Mol Sci. 2009 December; 10(12): 5442-5454; Buckley Am J Physiol Lung Cell Mol Physiol 288: L1132-L1138, 2005. U.S. Pat. No. 7,655,752 teaches TACE pro-domain and fragments thereof for the treatment of cancer and other diseases.